AIM To identify dominant microorganisms in root-filled teeth with apical periodontitis by Pan-PCRs in comparison to a culture-dependent approach, focusing on fungal species profiling. METHODOLOGY The root filling material (gutta-percha) removed from 42 teeth undergoing root canal treatment with periapical radiolucency was analysed by molecular genetics techniques. Real-Time Pan-PCRs were conducted for the diagnosis of predominant bacteria (targeting 16S rDNA) and fungi (targeting ITS1-2 region). Identification of microorganisms was performed by Sanger sequencing of the PCR products and BLAST analysis. Additionally, subgingival plaque samples were collected and cultured to review the microbial flora's composition. The McNemar test and the repeated measures ANOVA were used for statistical analyses (significance level was set at p<0.05). RESULTS Overall, 42/42 plaque samples had bacterial growth, whereas 32/42 gutta-percha samples had bacterial growth with a dominance of Streptococcus spp. (12/42) and Enterococcus faecalis (9/42). The mean number of bacterial taxa per gutta-percha sample was 1.6 cultivatable taxa, significantly lower than in the plaque sample with 6 taxa/sample (p<0.001). Fungus-specific cultures were negative for gutta-percha samples and only one plaque sample had growth of a fungus. In total, 36/42 plaque samples were positive in bacterial Pan-PCRs. In bacterial Pan-PCRs of 31/42 gutta-percha samples dominant microorganisms were identified including Streptococcus spp. (5/42) and E. faecalis (4/42). Moreover, in 7/42 gutta-percha samples DNA of bacteria which are difficult-to-cultivate in microbiology routine culture (Acinetobacter, Pyramidobacter, Bacteroidetes, Synergistes, Atopobium, Pseudoramibacter) were found. DNA of Candida spp. was detected in 5/42 root canals by fungal Pan-PCR (1/5) and genus-specific Candida-PCR (5/5). CONCLUSIONS Pan-PCR assays remain appropriate as a broad-range approach for the detection of a dominant pathogen in gutta-percha samples which have less diverse microbial composition. The molecular genetic Pan-PCR approach has the advantage of detecting microorganisms that are as-yet-uncultivable or difficult-to-cultivate and should be therefore complementary to conventional microbiological diagnostics.

中文翻译：

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独立于培养的方法在重新感染牙根的牙齿中检测细菌和真菌的好处-一项初步研究。

目的通过泛PCR（Pan-PCRs）技术与培养依赖方法（以真菌种类分析为重点）相比，鉴定根尖牙周炎的根部充填牙齿中的优势微生物。方法通过分子遗传学技术分析了从根尖不透性接受根管治疗的42颗牙齿中去除的牙根填充材料（牙胶）。进行实时Pan-PCR来诊断主要细菌（靶向16S rDNA）和真菌（靶向ITS1-2区）。通过PCR产物的Sanger测序和BLAST分析进行微生物鉴定。另外，收集并培养龈下菌斑样品以检查微生物菌群的组成。使用McNemar检验和重复测量ANOVA进行统计分析（显着性水平设置为p <0.05）。结果总体上，42/42菌斑样品具有细菌生长，而32/42牙胶样品具有细菌生长，且以链球菌属为主。（12/42）和粪肠球菌（9/42）。每个牙胶样品的细菌分类单元的平均数量为1.6个可耕种分类单元，显着低于菌斑样品的6个分类单元/样品（p <0.001）。特定于真菌的培养物对牙胶的样本阴性，并且只有一个噬菌斑样本具有真菌的生长。总共有36/42个噬菌斑样品在细菌Pan-PCR中呈阳性。在31/42牙胶样品的细菌Pan-PCR中，鉴定出主要的微生物，包括链球菌。（5/42）和粪肠球菌（4/42）。此外，在7/42的牙胶样品中，微生物常规培养中难以培养的细菌DNA（不动杆菌，金字塔形细菌，发现了拟杆菌，增效剂，拟南芥，假单胞杆菌。念珠菌属的DNA。通过真菌Pan-PCR（1/5）和属特异性念珠菌（Candida-PCR）（5/5）在5/42根管中检测到了TMP。结论Pan-PCR测定法仍然是适合检测微生物组成较少的牙胶样品中主要病原体的广泛方法。分子遗传Pan-PCR方法的优点是可以检测尚未培养或难以培养的微生物，因此应与常规微生物学诊断方法互补。结论Pan-PCR测定法仍然是适合检测微生物组成较少的牙胶样品中主要病原体的广泛方法。分子遗传Pan-PCR方法的优点是可以检测尚未培养或难以培养的微生物，因此应与常规微生物学诊断方法互补。结论Pan-PCR测定法仍然是适合检测微生物组成较少的牙胶样品中主要病原体的广泛方法。分子遗传Pan-PCR方法的优点是可以检测尚未培养或难以培养的微生物，因此应与常规微生物学诊断方法互补。